The overall goal of our research is to study the molecular basis of reverse transcriptase (RT)-mediated transposition in eukaryotes. We are analyzing this phenomenon in the insect trypanosomatid Crithidia fasciculata, a protozoan parasite which serves as host to the site- specific retrotransposons CRE1 and CRE2. CRE-elements are members of the little understood family of mobile genes known as non-LTR retrotransposons that are widely distributed in mammals, insects, plants, trypanosomatids, and fungi. Evidence suggests that this class of transposon is involved in such diverse processes as genome evolution, pseudogene formation, and human genetic disease initiation, yet almost nothing is known about the mechanisms by which these elements transpose. CRE1 and CRE2 are inserted at a specific highly conserved site in the tandemly arrayed miniexon genes. CRE1 rapidly rearranges within the C.fasciculata miniexon locus and encodes a novel RT activity. The purpose of this proposal is to extend and explore these observations, focusing on questions regarding the mechanisms underlying CRE1 genomic rearrangement and the extent of retrotransposon invasion of the Crithidia genome. Accomplishment of the specific aims will advance our knowledge of the mechanisms involved in the replication of this poorly understood but biologically important class of retrotransposons. Characterization of genetic events occurring within the miniexon gene locus has potential therapeutic implications for both African sleeping sickness and Chagas' disease. On a broader scale, the results will be relevant to questions concerning eukaryotic genome organization and evolution, as well as the pathogenesis of human genetic disease. We propose to: (1) Characterize the RT activity encoded by CRE1 both in terms of its functional domains and its primer and template specificity, using both a protein expression system that targets fusion proteins to yeast Ty1 virus-like particles (VLPs) and a genetic assay that results in CRE1 RT-mediated pseudogene formation in yeast; (2) Identify and characterize additional site-specific retrotransposons inserted into the C.fasciculata miniexon array; (3) Determine the genomic organization of CRE1 and related site-specific retrotransposons in the Crithidia miniexon array, and dissect the genetic mechanisms, including site-specific transposition and homologous recombination, by which CRE1 rearranges within the genome of C.fasciculata.